WO2010010223A1 - Use of microparticles containing genetically modified cells in the treatment of neurodegenerative diseases - Google Patents

Abstract

The invention relates to methods for the treatment of neurodegenerative diseases, comprising the use of microparticles containing genetically modified cells expressing neurotrophic and/or angiogenic factors, in which said microparticles are administered by means of implantation into the cerebral cortex.

Description

 EMPLOYMENT OF MICROPARTURES THAT INCLUDE CELLS

GENETICALLY MODIFIED IN THE TREATMENT OF

NEURODEGENERATIVE DISEASES

FIELD OF THE INVENTION

The present invention relates to the use of microparticles comprising genetically modified cells that express at least one neurotrophic factor, at least one angiogenic factor or a combination of both for the treatment of neurodegenerative diseases.

BACKGROUND OF THE INVENTION

Neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), among others, constitute a serious problem from the medical, care, social and economic point of view that developed countries must face .

There are currently several medications that provide symptomatic treatment of these diseases, but none that have proven useful with clinical relevance to slow the progression of the degenerative process.

Angiogenic factors are growth factors that not only stimulate neovascularization and angiogenesis (initiated with the activation of endothelial cells of parental blood vessels) in vivo, but are also mitogenic for endothelial cells in vitro. Examples of angiogenic factors are, for example, HGF, VEGF, FGF and HIF. VEGF (vascular endothelial growth factor) is the prototype of angiogenic factors, whose role at the level of the Central Nervous System (CNS) is not only related to the growth of blood vessels, as a physiological regulator of cerebral angiogenesis and the integrity of the blood brain barrier, but also has a direct effect on different types of neuronal cells, including even neuronal stem cells (NSCs). On the other hand, studies show that cerebrovascular deficiencies occur in diseases such as AD or Huntington that They precede the onset of clinical symptoms, which suggests that such changes could contribute to the pathogenesis of these diseases.

Neurotrophic factors are natural proteins that play an important role at the CNS level. These factors are essential to ensure the survival and differentiation of neurons during development and to maintain normal neuronal function in the adult. Due to these physiological functions, neurotrophic factors are useful for the treatment of CNS pathologies in which survival and / or the neuronal function itself are compromised. Among the neurotrophic factors is GDNF (glia-derived neurotrophic factor) that was isolated by Lin and cois in 1930 and since then its use has been tested in different diseases that affect the CNS.

An important problem to solve is the system and the place of administration of these angiogenic and neurotrophic factors, since it is necessary that the factor crosses the blood-brain barrier so that it can exert its action and also the treatment of these diseases can lead to an administration chronic for long periods of time.

Based on previous knowledge, a large number of therapeutic products are available, such as, for example, the neurotrophic and angiogenic factors mentioned above, for the treatment of CNS diseases. However, the release of these products at the brain level is limited by the difficulty of accessing the site of administration. In clinical practice, the controlled release of these therapeutic products is performed by infusion pumps or cannulas at the intraventricular level. These routes of administration require continuous injections or refills of the infusion pump to maintain drug levels and prevent degradation of the therapeutic substance. In addition, due to the technology presented by current infusion pumps, it is difficult to administer low doses of drugs for prolonged periods of time.

One of the therapeutic strategies that is becoming more important in the treatment of neurodegenerative diseases and CNS is cell therapy. The The use of cells as a medicine is a therapeutic alternative of growing interest in the scientific community, with which it is intended to develop pharmaceutical systems that release the drug secreted by the cells for long periods of time and in a safe and physiological way, being especially suitable for Treatment of chronic diseases. However, one of the main limitations of this type of therapy is the rejection by the host's immune response to the implanted cells, as long as the transplant graft does not come from the host itself. Hence, the administration of allogeneic and xenogeneic cells secreting therapeutic products, offers unsatisfactory results in the medium to long term as a result of the action of the patient's defensive mechanisms. Due to this disadvantage, systems have been designed that allow the administration of cells and that avoid the rejection caused by the immune response, among these systems, hollow fibers and microcapsules stand out.

The use of hollow fibers containing genetically modified cells to produce CNTF (Ciliary neurotrophic factor) is an administration system that has been used in the treatment of patients with ALS. Likewise, procedures have also been described which include the encapsulation of GDNF or VEGF producing cells in hollow fibers and implanted at the striatum level (Yasuhara, T. and Date, I. 2007. CeIl transplantation, vol. 16: 1-8; Lindvall, O. and Wahlberg, LU 2008, Experimental Neurology, vol. 209: 82-88). European patent application EP0388428 describes methods in which ventral mesencephalon fragments encapsulated in tubes formed by semipermeable membranes are implanted in the parietal region of the cerebral cortex after craniotomy. However, this type of technology has the disadvantage of being a system of a large size (of the order of millimeters), which can condition the viability and functionality of the encapsulated cells.

An alternative to hollow fibers is the use of microcapsules that can comprise cells or fragments of the choroid plexus, or genetically modified cells to express proteins of therapeutic interest. Skinner, SMJ et al. (Xenotransplantation, 2006 vol.13: 284-288) describe the use of microencapsulated choroid plexus cells in animal models of different CNS diseases.

Borlongan et al. (Neurochemistry Int. 2004 vol. 24: 495-503) describe a method for the treatment of cerebral ischemia by subdural implantation of microcapsules comprising choroid plexus cells.

US patent application US2004213768 describes methods for the administration of neurotrophic factors faithful to the central nervous system based on the implantation of microcapsules (preferably alginate) comprising cells isolated from the choroid plexus, where the implantation is carried out in a subdural or subarachnoid manner.

However, the use of choroid plexus cells does not allow an exact control of the nature of the angiogenic / neurotrophic factors produced by the cell.

On the other hand, regarding the use of microcapsules that can comprise genetically modified cells, Meysinger, D. et al. (Neurochem. Int. 1994 vol. 24 (5): 495-503) describe alginate-polylysine-alginate microcapsules comprising genetically modified fibroblasts to produce nerve growth factor (NGF). In a work published by Grandoso, L. et al. (Internal Journal of Pharmaceutics, 2007 vol. 343: 69-78) the use of alginate-poly-L-lysine microcapsules in which GDNF-producing fibroblasts for the treatment of PD in a parkinsonized rat model is studied . The microcapsules were administered at the striatum level by sterotaxy and an improvement in the rotational behavior of the animals was observed.

However, both works employ overly invasive techniques that can lead to unwanted side effects in the patient.

Therefore, there is a need in the state of the art to find systems that allow to control the therapeutic product and / or the angiogenic and neurotrophic factors administered and that are less invasive, thereby improving the treatment of neurodegenerative diseases, including AD and PD.

SUMMARY OF THE INVENTION In one aspect, the invention relates to a microparticle comprising genetically modified cells that express at least one neurotrophic factor, at least one angiogenic factor or a combination of both for the treatment of neurodegenerative diseases, wherein the administration of The microparticle is made at the level of the cerebral cortex.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows three photographs of microparticles containing genetically modified fibroblasts to produce VEGF. A and B: optical microscopy images. C: fluorescence microscopy image.

Figure 2 is a graph showing the proliferation of BMVEC cells in the presence of VEGF secreted by the microparticles (33 ng / mL) and the stock solution of VEGF (500 ng / mL) on days 7 and 20.

Figure 3 is a graph showing the density of blood vessels stimulated by the production of VEGF from microparticles implanted in normal C57BL / 6 mice.

Figure 4 is a photograph showing the double BrdUrd-lectin staining of new blood vessels induced by the production of VEGF from microparticles implanted in normal C57BL / 6 mice.

Figure 5 is a graph representing the results obtained in the T-maze test in APP / PS1 mice.

Figure 6 is a graph showing the results of the object recognition test in APP / PS1 mice. Figure 7 is a graph depicting the levels of BDNF measured in different tissues of old (18-24 month) control rats (treated empty microparticles) and rats treated with microparticles containing BDNF producing cells.

Figure 8 is a graph depicting the results of the determination of caspase 3, caspase 9 and PMAPK in cortex of old control rats (treated with empty microparticles) and with the microparticles that release BDNF.

DETA CALL DESCRIPTION OF THE INVENTION The inventors have discovered that the administration at the level of the cerebral cortex of microparticles comprising genetically modified cells to express neurotrophic or angiogenic factors allows, surprisingly, the treatment not only of lesions located in the cerebral cortex, but also lesions located in other areas of the brain thanks to the diffusion of the neurotrophic or angiogenic factors administered.

Thus, in one aspect, the invention relates to a microparticle comprising genetically modified cells that express at least one neurotrophic factor, at least one angiogenic factor or a combination of both, for the treatment of neurodegenerative diseases, wherein the administration of the Microparticle is made at the level of the cerebral cortex.

The microparticle of the invention is a spherical particle or not, which includes (i) microcapsules, which are defined as vesicular systems in which genetically modified cells are confined in a cavity surrounded by a single membrane (usually polymeric) ; and (ii) microspheres, which are matrix systems in which cells are dispersed throughout the particle.

In the present invention, "microparticle" is understood as that particle comprising a diameter of less than 1 mm, preferably between 1 and 0.9, between 0.9 and 0.8, between 0.8 and 0.7, between 0.7 and 0.6, between 0.6 and 0.5, between 0.5 and 0.4, between 0.4 and 0.3, between 0.3 and 0.2, between 0.2 and 0.1 or less than 0.1 mm in diameter. In a particular embodiment, the microparticle of the invention has a diameter between 0.380 and 0.404 mm, preferably 0.392 mm. Also, in the context of the present invention and due to the ability of the microparticle to continuously produce therapeutic products, said microparticle is called the pharmaceutical micro-biosystem.

However, as the person skilled in the art understands, the average size of the microparticle of the invention is influenced by different technological factors of the method of producing said microparticle, such as the concentration of the different components of the microparticle, stirring speed. , etc.

The microparticle of the invention can be formed by any biocompatible polymeric material that allows the continuous secretion of the therapeutic products and that acts as a support for the genetically modified cells. Thus, said biocompatible polymeric material can be, for example, thermoplastic polymers or hydrogel polymers.

Among the thermoplastic polymers are acrylic acid, acrylamide, 2-aminoethyl methacrylate, poly (tetrafluoroethylene-cohexafluoropropylene), methacrylic acid- (7- cumaroxy) ethyl ester, N-isopropyl acrylamide, polyacrylic acid, polyacrylamide, polyamidoamia -p-xylylene, poly (chloroethyl vinyl ether), polycaprolactone, poly (caprlactone-co-trimethylene carboanto), poly (carbonate urea) urethane, poly (carbonate) urethane, polyethylene, polyethylene and archylamide co-polymer, polyethylene glycol, polyethylene glycol methacrylate ethylene terephthalate), poly (4-hydroxybutyl acrylate), poly (hydroxyethyl methacrylate), poly (N-2-hydroxypropyl methacrylate), poly (lactic acid-glycolic acid), poly (lactic acid L), poly (gamma-methyl), L-glutamate), poly (methyl methacrylate), polypropylene fumarate), polypropylene oxide), polypyrrole, polystyrene, poly (tetrafluoro ethylene), polyurethane, polyvinyl alcohol, ultra high molecular weight polyethylene, 6- (p-vinylbenzamido) -hexan acid oico and N-p-vinylbenzyl-D-maltonamide and copolymers containing more than one of said polymers.

Among the polymers of the hydrogel type are natural materials such as alginate, agarose, collagen, starch, hyaluronic acid, bovine serum albumin, cellulose and its derivatives, pectin, chondroitin sulfate, fibrin and fibroin, as well as synthetic hydrogels such as sepharose and sephadex . In a particular embodiment, the polymer that is part of the microparticle of the invention is linked to, or functionalized with, a specific ligand for a cell surface receptor. In the present invention, "specific ligand for a cell surface receptor" is understood to be the molecule or peptide that is capable of recognizing a cell surface receptor and specifically binding to it. Thus, said ligand allows the specific interaction between the polymer of the microparticle and the genetically modified cells contained within it.

In principle, any molecule or peptide that possesses specific binding sites that can be recognized by cell surface receptors can be used in the present invention as a specific ligand for a cell surface receptor.

Thus, the specific ligand for a cell surface receptor can come from cell adhesion molecules that interact with the extracellular matrix such as fibronectin, the different members of the selectin families, the caderins, lectins, integrins, immunoglobulins, the collective and the galectinas.

Thus, the specific ligand for a cell surface receptor employed in the present invention can be a peptide derived from a region selected from the fibronectin regions that are involved in binding with the integrins found in the cell membrane. For example, and without limiting the invention, said peptides are derived from the region of the tenth repetition type III of fibronectin containing the RGD peptide, from the region of the fourteenth repetition type III of the fibronectin containing the peptide IDAPS (SEQ ID NO : 1), of the CSI region of fibronectin containing the LDV peptide and the CS5 region of fibronectin containing the REDV peptide (SEQ ID NO: 2). These peptides may consist of fragments of the corresponding regions that retain their adhesive capacity, such as, for example, the QAGDV peptide (SEQ ID NO: 3) of the fibrinogen, the LDV peptide of the fibronectin and the peptide IDSP (SEQ ID NO: 4 ) of VCAM-I.

The present invention also contemplates the use of integrin binding peptides as a specific ligand for a cell surface receptor, which are derived from the region of the tenth type III fibronectin repeat comprising the RGD sequence, such as, for example, a peptide selected from the group of RGD, RGDS (SEQ ID NO: 5), GRGD (SEQ ID NO: 6), RGDV (SEQ ID NO: 7), RGDT (SEQ ID NO: 8), GRGDG (SEQ ID NO: 9), GRGDS (SEQ ID NO: 10), GRGDY (SEQ ID NO : 11), GRGDF (SEQ ID NO: 12), YRGDS (SEQ ID NO: 13), YRGDDG (SEQ ID NO: 14), GRGDSP (SEQ ID NO: 15), GRGDSG (SEQ ID NO: 16), GRGDSY (SEQ ID NO: 17), GRGDVY (SEQ ID NO: 18), GRGDSPK (SEQ ID NO: 19), CGRGDSPK (SEQ ID NO: 20), CGRGDSPK (SEQ ID NO: 21), CGRGDSY (SEQ ID NO: 22), cycle (RGDfK) (SEQ ID NO: 23), YAVTGRGD (SEQ ID NO: 24), AcCGGNGEPRGDYRAY-NH2 (SEQ ID NO: 25), AcGCGYGRGDSPG (SEQ ID NO: 26) and RGDPASSKP (SEQ ID NO: 27), cyclic variants of said peptides, both linear and branched multivalent variants (see for example Dettin et al. 2002, J. Biomed. Mater. Res. 60: 466-471; Monaghan et al. 2001, Arkivoc, 2: U42 -49; Thumshirn et al. 2003, Chemistry 9: 2717-2725; Scott et al., 2001, J. Gene Med. 3: 125-134) as well as combinations of two or more of said peptides.

Therefore, in another particular embodiment, the specific ligand for a cell surface receptor is a peptide comprising the RGD sequence.

The peptide comprising the RGD sequence may be attached to the polymer of the microparticle through the N-terminal or C-terminal end and, regardless of the anchor point, may be directly attached to the polymer or, alternatively, may be attached to through a spacer element. Virtually any peptide with structural flexibility can be used. By way of illustration, said flexible peptide may contain amino acid residue repeats, such as (GIy) ₄ (SEQ ID NO: 28), Gly-Gly-Gly-Ser (SEQ ID NO: 29), (GIy) _n (SEQ ID NO: 30) (Beer, JH et al, 1992, Blood, 79, 117-128), SGGTSGSTSGTGST (SEQ ID NO: 31), AGSSTGSSTGPGSTT (SEQ ID NO: 32), GGSGGAP (SEQ ID NO: 33), GGGVEGGG (SEQ ID NO: 34) or any other suitable amino acid residue repeat, or the hinge region of an antibody.

Also, the specific ligand for a cell surface receptor can be bound to the polymer with varying degrees of substitution, so that the concentrations of both components can vary and thus control the number of specific ligands for a cell surface receptor that are bound to the polymer of the microparticle Thus, the invention contemplates polymers containing between 1 and 100, between 100 and 200, between 200 and 300, between 300 and 400, between 400 and 500, between 500 and 600, between 600 and 700, between 700 and 800, between 800 and 900 and between 900 and 1000 molecules of said specific ligand for each polymer molecule.

As indicated above, the microparticle of the invention can be formed by any biocompatible polymeric material that allows the continuous secretion of the therapeutic products and acts as a support for the genetically modified cells. Thus, in a particular embodiment, the polymer of the microparticle of the invention is alginate. In Example 1 of the present patent application one of the different methods that exist in the state of the art to produce microparticles comprising alginate as a biopolymeric material is described.

In principle, any type of alginate capable of forming a hydrogel is suitable for use in the microparticle of the invention. Thus, the microparticle can contain alginate formed mostly by manuronic acid regions (MM blocks), by guluronic acid regions (GG blocks) and by mixed sequence regions (MG blocks). The percentage and distribution of uronic acids differ according to the origin of the alginate and contribute to the properties of the alginate. The person skilled in the art knows the percentages of each of the different blocks that appear in the different biological sources of alginates. Thus, the invention contemplates the use of alginates from Laminaria hyperborea, Latvia nigrescens, Lessonia trabeculata, Durvillaea antarctica, Laminaria digitata, Eclonia maxim, Macrocystis pyrifera, Ascophyllum nodosum and / or Laminaria japanica as well as mixtures of alginates of different species until the desired content in GG, MM or GM blocks. The GG blocks contribute to the rigidity of the hydrogel, while the MM monomers maintain a high resistance to fracture, so that by using a suitable combination of alginate polymers, a mixture whose modulus of elasticity has a value can be obtained adequate while the viscosity of the pre-gel solution is maintained at sufficiently low levels to allow adequate cell manipulation and immobilization. Thus, the alginates that can be used in the present invention include GG alginates, MM alginates or combinations of both in a ratio of 90: 10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80 or 10:90.

Additionally, the invention also contemplates the use of alginates derived from the treatment of natural alginates with enzymes that are capable of modifying the integral blocks to give rise to alginates with improved properties. Thus, alginates resulting from treating alginates with C5-epimerases, which convert M blocks into G blocks, as well as with the AlgE4 enzyme of the bacterium Azotobacter vinelandii which is capable of converting the relatively rigid M blocks into MG blocks. Alternatively, the invention contemplates the use of alginates that have been modified by different physical treatments, in particular, gamma rays, irradiation with ultrasound or with ultraviolet light as described by Wasikiewicz, J. M. et al. (Radiation Physics and Chemistry, 2005, 73: 287-295).

The microparticle of the invention comprising alginate as a biocompatible polymeric material can be used as is. However, as is known from the state of the art, alginate is a poorly stable polymer that tends to lose calcium and therefore, lose its gel character. In addition, alginate particles are relatively porous which results in antibodies being able to access inside and damage cells. For these reasons, optionally, the microparticle of the invention may be surrounded by a semipermeable membrane that confers stability to the particles and forms a barrier impermeable to antibodies.

Semi-permeable membrane means a membrane that allows the entry of all those solutes necessary for cell viability and that allows the therapeutic proteins produced by the cells contained within the microparticle to be exited, but which is substantially impermeable to antibodies, so that the cells are protected from the immune response produced by the organism that hosts the microparticle.

Suitable materials for forming the semipermeable membrane are materials insoluble in biological fluids, preferably polyamino acids, such as poly-L-lysine, poly-L-ornithine, poly-L-arginine, poly-L-asparagine, poly-L-aspartic cop benzyl-L-aspartate, poly-S-benzyl-L-cysteine, poly-gamma-benzyl-L-glutamate, poly-S-CBZ-L-cysteine, poly-ε-CBZ-D-lysine, poly-δ- CBZ-DL-Ornithine, poly-O-CBZ-L-serine, poly-O-CBZ-D-tyrosine, poly (γ-ethyl-L-glutamate), poly-D-glutamic, polyglycine, poly-γ- N -hexyl L-glutamate, poly-L-histidine, poly (α, β- [N- (2-hydroxyethyl) -DL-aspartamide]), poly-L-hydroxyproline, poly (α, β- [N- (3 -hydroxypropyl) -DL-aspartamide]), poly-L-isoleucine, poly-L-leucine, poly-D-lysine, poly-L-phenylalanine, poly-L-proline, poly-L-serine, poly-L- threonine, poly-DL-tryptophan, poly-D-tyrosine or a combination thereof. Preferably,

Therefore, in a particular embodiment, the microparticle of the invention further comprises a poly-L-lysine membrane.

The membrane that covers the microparticle is usually of a polycationic material, which results in the formation of a polyanion-polycation complex that contributes to the stabilization of the alginate and to reduce the porosity of the microparticle and to form an immune barrier that is impervious to antibodies. However, the positive charge of said membrane favors cell adhesion to the surface of the microparticle resulting in a lower biocompatibility thereof. Therefore, if desired, the poly-L-Lysine membrane that surrounds the microparticle is, in turn, surrounded by a second membrane formed mostly of a material that inhibits cell adhesion, preferably alginate (see Example 1 of the present patent application).

Any eukaryotic cell that has been genetically modified to express at least one neurotrophic factor, at least one angiogenic factor or a combination of both, can be used in the present invention, but the mouse, rat, primate and human cells are the preferred ones. Thus suitable cells for carrying out the invention are cardiomyocytes, endothelial cells, epithelial cells, lymphocytes (B and T cells), mast cells, eosinophils, vascular intimal cells, primary cultures of isolated cells of different organs, preferably isolated cells of the islets of Langerhans, hepatocytes, leukocytes, including mononuclear leukocytes, stem cells of embryonic origin, mesenchymal, umbilical cord or adult (skin, lung, kidney and liver), osteoclasts, chondrocytes and other tissue cells connective. Also suitable are cells of established lines such as Jurkat T cells, NIH-3T3, CHO, Cos, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, C2C12 myoblasts and W138 cells.

In principle, the number of cells that must be part of the microparticle is not essential for the invention as long as there is a sufficient number of cells to contribute to the formation of the lattice. Thus, the amount of cells per mL of polymer solution is between 1 and 10 x ^{6 6} , preferably between 2 and 9 x ⁶ , more preferably between 3 and 8 x ⁶ , even more preferably between 4 and 7 x ⁶ and even more preferably between 5 and 6 x ^{6 6} . Preferably, the number of cells in the initial mixture is 5; 3.75; 2.5 or 1.25 xl O ⁶ per mL of polymer solution.

In the context of the present invention, the eukaryotic cell contained within the microparticle of the invention can be modified with any neurotrophic factor, for example, and not limited to, neurotrophins (NT), such as NT-3, NT-4, NT-5 or NT-6; Brain-derived neurotrophic factor (BDNF, Brain-derived neurotrophic factor); ciliary neurotrophic factor (CNTF); insulin growth factor type 1 (IGF-I); insulin growth factor type 2 (IGF-2); Nerve Growth Factor (NGF); neurturin (NTN); persefinas; artemines, pleiotrophin (PTN), efrines, netrins, semaphoreins, slits, reelins, glia-derived neurotrophic factor (GDNF), conserved dopamine neurotrophic factor (CDNF) (conserved dopamine neurotrophic factor), mesothalic astrocyte-derived neurotrophic factor ( MANF, mesencephalic astrocyte-derived neurotrophic factor) etc.

Also, in the context of the present invention, the eukaryotic cell contained within the microparticle of the invention can be modified with any angiogenic factor, for example, and not limited to, angiopoietin (Ang), such as Ang-1, Ang- 2, Ang-3 or Ang-4; basic fibroblast growth factor (bFGF / FGF2), such as fibroblast growth factor 20 (FGF20); transforming growth factor beta (TGFβ), transforming growth factor alpha (TGFα), placental growth factor (PIGF); epidermal growth factor (EGF, epidemic grothw factor); vascular endothelial growth factor (VEGF), such as VEGF-A, VEGF-B or VEGF-C; platelet derived growth factor (PDGF, Platelet-derived growth factor), such as PDGF-A and PDGF-B; vasoactive intestinal polypeptide (VIP); hepatocyte growth factor (HGF), cardiotrophins, bone morphogenetic proteins (BMP), sonic hedgehog (SHH), etc.

As the person skilled in the art understands, the genetic modification of the cells to be encapsulated within the microparticle of the invention can be carried out by any method known in the art. Thus, the gene or the vector containing the gene can be administered by electroporation, transfection using liposomes or polycationic proteins or using viral vectors, including adenoviral and retroviral vectors and also non-viral vectors.

Likewise, the nucleotide sequences that encode neurotrophic factors or angiogenic factors are associated with sequences that regulate the expression of said factors. These sequences can be sequences that regulate transcription, as promoters, constitutive or inducible, enhancers, transcriptional terminators and sequences that regulate translation, such as untranslated sequences located 5 'or 3' with respect to the coding sequence.

Suitable promoters for the expression of neurotrophic factors or angiogenic factors include, but are not necessarily limited to, constitutive promoters such as those derived from eukaryotic virus genomes such as polyoma virus, adenovirus, SV40, CMV, avian sarcoma virus, hepatitis B virus, the metallothionein gene promoter, the herpes simplex virus thymidine kinase gene promoter, retrovirus LTR regions, the immunoglobuin gene promoter, the actin gene promoter, the promoter of the EF-I alpha gene as well as inducible promoters in which protein expression depends on the addition of a molecule or an exogenous exogenous signal, such as the tetracycline system, the NFkappaB / UV light system, the Cre system / Lox and the heat shock gene promoter.

On the other hand, neurotrophic factors or angiogenic factors expressed by the cells that are part of the microparticle of the invention can be expressed as temporarily or stably. In the event that the microparticle remains a prolonged time in the patient, it is preferable to use cells that express said factors stably. Stable expression requires the transformation of the polynucleotide that encodes neurotrophic factors or angiogenic factors be performed in conjunction with a polynucleotide that encodes a protein that allows to select transformed cells. Suitable selection systems are, without limitation, the herpes virus thymidine kinase, the phosphoribosyltransferase hypoxanthine guanine, adenine phosphoribosyltransferase, genes encoding proteins that confer resistance to an antimetabolite such as dihydrofolate reduced, pyruvate transaminase, the resistance gene to Neomycin and hygromycin.

As indicated at the beginning of the present description, the invention relates to a microparticle comprising genetically modified cells that express at least one neurotrophic factor, at least one angiogenic factor or a combination of both, for the treatment of neurodegenerative diseases, where the administration of the microparticle is performed at the level of the cerebral cortex.

As the person skilled in the art understands, any neurodegenerative disease that can be treated with neurotrophic factors, angiogenic factors or a combination of both, can also be treated with the microparticle of the invention.

In the present invention, "neurodegenerative disease" is understood as that disease that is distinguished by being the result of a progressive death of neurons in the nervous system, primarily in the brain, resulting in the worsening of bodily activities, such as balance , movement, speech, breathing, cardiac function, etc. Examples of neurodegenerative diseases are, without limitation, Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, dementia with Lewy bodies, Parkinson's disease, spinal muscular atrophy, etc.

In a particular embodiment, neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and Huntington's disease. In the context of the present invention, administration of the microparticle is performed at the level of the cerebral cortex, allowing the diffusion of neurotrophic or angiogenic factors to other areas of the brain. By administration "at the level of the cerebral cortex" means, in the context of the present invention, a method in which the administration is carried out by perforation of the skull and one or several meninges but without damaging the cerebral cortex.

In the context of the present invention, the preferred method of administration is by craniotomy.

In the present invention, "craniotomy" is understood as surgery of the brain consisting of the opening of the skull to expose the meninges, allowing the administration of the microparticles in a subdural or subarachnoid manner.

In a particular embodiment of the invention, administration of the microparticle at the level of the cerebral cortex is carried out in a subdural or subarachnoid manner. The procedure for administering the microparticles is described in Example 2 that accompanies the present description.

In the present invention, "cerebral cortex" or "cerebral cortex" is understood as the mantle of nerve tissue that covers the surface of the cerebral hemispheres.

In a particular embodiment, administration of the microparticle at the level of the cerebral cortex is carried out by bilateral craniotomy.

In another aspect, the invention relates to a method for the treatment of neurodegenerative diseases by administering microparticles comprising genetically modified cells that express at least one neurotrophic factor, at least one angiogenic factor or a combination of the foregoing, wherein the Administration of the microparticles is done at the level of the cerebral cortex.

The following Examples are illustrative of the invention and are not intended to be limiting thereof. EXAMPLE 1

Development and characterization of microparticles

Cultivation of VEGF producing cells

BHK cells (fibroblasts from hamster kidney) genetically modified to produce VEGF were grown in DMEM medium with 2% L-glutamine, 10% fetal bovine serum (FBS) and 1% antibiotic / antifungal. They pass the cells every 2 or 3 days were made, remaining in the incubator at 37 ⁰ C in an atmosphere of 5% CO _2. All components of the culture media used were from the Gibco BRL house (Invitrogen SA, Spain).

Encapsulation of the cells in the microparticles

The encapsulation process comprises several stages. First, the cells, genetically modified to secrete the therapeutic product, are suspended in an alginate solution. Subsequently, the cell suspension is passed through the tip of the electrostatic dripper by means of a flow pump, the droplets formed falling into the gelling solution of calcium chloride. Furthermore, by applying a difference in electrostatic potential between the drip tip and the calcium chloride solution, the formation and gelation of small droplets from the cell suspension is achieved. Once the solid alginate cores are formed, they are coated, applying a first coating with a 0.05% poly-L-lysine solution for 5 minutes and a second coating with a 0.1% alginate solution for 5 minutes.

Characterization of the microparticles

The size and surface characteristics of the microparticles were determined using an inverted optical microscope (NikonTSM) equipped with a camera (Sony CCD-Iris). The microparticles obtained are spherical in shape and have a smooth and uniform surface whose average size was 392 ± 12 μm (Figure 1). Viability and functionality of VEGF producing fibroblasts in microparticles

To characterize the viability and functionality of the fibroblasts inside the microparticles, the cellular metabolic activity and the release of VEGF were studied in vitro over a period of three weeks. Cell viability was determined using the MTT assay. VEGF production was determined using an ELISA technique (Amersham Biosciences, USA). After 21 days in culture, the secretion of VEGF from the microparticles loaded with 10 ⁶ cells per milliliter of alginate was approximately 174 ng of VEGF / 24 hours. These results suggest that the cells successfully adapted to the new micro environment.

In vitro proliferation assay

The BMVEC cell line are cells that are derived from brain endothelial cells. They are maintained in DMEM medium (Sigma), 10% FBS (Gibco) and 1% antifungal antibiotic (Gibco).

BMVEC brain endothelial cells were cultured in the presence of different concentrations of VEGF prepared from the pure product, stock solution (500 ng / mL) and secreted from the microparticles, functional VEGF (33 ng / mL). Cell proliferation was determined by the XTT Proliferation Kit (Roche) at 7 and 20 days (Figure 2). This test allows to determine the functionality of the VEGF released from the microparticles. The concentration of 33 ng / mL is similar to that produced by about 200 microparticles and its effect was compared with a high concentration of VEGF such as 500 ng / mL. The results obtained show that at 7 days both the treatment with the stock solution and with the functional VEGF stimulate cell proliferation and that this is significantly superior to that produced by the controls. Surprisingly at 20 days it is observed that low concentrations of VEGF induce a higher cell proliferation than high concentrations, which shows that the dose of VEGF secreted by the microparticles could be sufficient to induce the proliferative response without any risk of occurrence of edema caused by an excessively high concentration of VEGF. EXAMPLE 2

Implantation of microparticles in normal C57BL / 6 mice and histological analysis

The animals were divided into two groups of 6 mice each: control (received empty microparticles) and treated with VEGF (n = 6) to which microparticles containing VEGF-producing cells were implanted at the level of the cerebral cortex . Mice anesthetized by inhaled isofluorane had a bilateral craniotomy at the posterior 0.6 mm and 1.1 mm lateral coordinates at the bregma point (Paxinos & Watson, 1986. "The rat brain in stereotaxic coordinates", 2nd edition, New York : Academy Once the craniotomy was performed, VEGF-producing microparticles were administered on the surface of the brain and empty microparticles were administered to the control mice, each mouse was administered between 20-30 microparticles per hole. Surgically, the holes were closed with nitrocellulose membranes impregnated in a dilute iodine solution (Betadine), which prevents the capsules from leaving the holes and isolates the brain from a possible infection.

The animals were sacrificed at 2 weeks, 1 month and 3 months and immunohistochemical studies were performed. Vessel formation was determined using biotinylated tomato lectin and factor VIII.

The results obtained showed that the density of blood vessels in the brains of animals treated with microparticles containing VEGF-producing cells was clearly higher than that presented by animals in the control group at two weeks, and that the effect was maintained at one month and two months, which indicates a sustained release of the molecule from the microparticles (Figure 3).

In addition, double staining was performed with BrdUrd and lectin in order to distinguish the new vessels formed after the release of VEGF from the pre-existing vessels, with an increase in these vessels as time elapses since administration (Figure 4) . EXAMPLE 3

Treatment of Alzheimer's disease transgenic animals with microparticles in which genetically modified cells have been encapsulated to produce VEGF

Double transgenic mice: Amyloid precursor protein / presenilin-1 (APP / Psl) resulting from the cross between the Tg2576 mouse (overexpresses the human APP695 protein) and the mutant mouse (M 146L mouse) for presenilin-1 (PsI). These mice are a model of amyloidosis for Alzheimer's disease.

Mice anesthetized by inhaled isofluorane had a bilateral craniotomy at the posterior coordinates 0.6 mm and lateral 1.1 mm at the bregma point (Paxinos and Watson, 1982). Once the craniotomy was performed, they were administered on the surface of the brain at a subdural level, to a group of mice, VEGF-producing microparticles and to another group of mice, microparticles with non-transfected fibroblasts (this group of mice were called "sham" ). In addition, a group of control littermate mice was included. Each mouse (6 per group) was administered between 20-30 microparticles per hole. Once the surgery was completed, the holes were closed with nitrocellulose membranes impregnated in a dilute iodine solution (Betadine), which prevents the microparticles from leaving the holes and isolates the brain from a possible infection

The mice were kept for 3 months in the animal farm until they were tested for behavior: T-Maze (T labyrinth) and object recognition. At the end of the behavioral tests, the mice were perfused transcardiacally with 0.9% saline. Half of the brain was fixed with 4% paraformaldehyde in 0.1 M saline solution pH 7.4, and the other half was frozen at -8O ⁰ C for subsequent biochemical tests.

In T-maze, which measures exploratory activity, there is a recovery in latency or decision time (Figure 5). The rate of spontaneous alternation is also recovered: 63% in the control group, 50% in mice treated with microparticles with non-transfected fibroblasts ("sham"), and 61% in the group of mice treated with microparticles with fibroblasts producing VEGF In the object recognition test, which measures short-term memory, there is also a significant recovery (Figure 6).

EXAMPLE 4

Treatment of old rats with microparticles in which genetically modified cells have been immobilized to produce BDNF

Wistar rats between 24 and 30 months were used to carry out this experiment, conducting a pilot study with control rats and treated rats. Genetically modified cells to produce BDNF were encapsulated using the same methodology as in Example 1. The microparticles were implanted in the rats by bilateral craniotomy. Control rats were treated in the same manner but were given empty microparticles. The rats remained in the stable until their sacrifice, at 2 months, to carry out the appropriate analyzes.

The results shown in Figure 7 show that the BDNF released from the microparticles, administered on the surface of the brain, is transported to the blood, following the cortex, hippocampus, plexus-choroid, cerebrospinal fluid and blood route.

Histochemical immunoassay to detect caspase 3, caspase 9 and MAPK in different tissues shows that the rats treated with the BDNF producing microparticles show, at the cortex level (Figure 8) and at the cerebellum level, a decrease in caspase 3 activity and caspase 9 which would indicate a lower cell death. In addition, it can be seen how an increase in MAPK activity occurs, which indicates the activation of BDNF signaling in this region. In the choroid plexuses it is observed that there are no variations in the levels of BDNF although there is a decrease in caspase 3 levels which could indicate a lower deterioration of the blood brain barrier.

Claims

1. A microparticle comprising genetically modified cells that express at least one neurotrophic factor, at least one angiogenic factor or a combination of both for the treatment of neurodegenerative diseases, wherein the administration of the microparticle is performed at the level of the cerebral cortex.

2. Microparticle according to claim 1, wherein the microparticle comprises a polymer bound to a specific ligand for a cell surface receptor.

3. Microparticle according to claim 2, wherein the specific ligand for a cell surface receptor is a peptide comprising the RGD sequence.

4. Microparticle according to claim 2 or 3, wherein the polymer that is part of the microparticle is alginate.

5. Microparticle according to claim 4, wherein the microparticle further comprises a poly-L-Lysine membrane.

6. Microparticle according to any one of claims 1 to 5, wherein the neurotrophic factor is selected from neurotrophins (NT); brain-derived neurotrophic factor (BDNF); ciliary neurotrophic factor (CNTF); insulin growth factor type 1 (IGF-I); insulin growth factor type 2 (IGF-2); nerve growth factor (NGF); neurturin (NTN); persefinas; artemines, pleiotrophin (PTN), efrins, netrins, semaphoreins, slits, reelins or glia-derived neurotrophic factor

(GDNF), preserved neurotrophic dopamine factor (CDNF) (conserved dopamine neurotrophic factor) and neurotrophic factor derived from mesencephalic astrocytes (MANF, mesencephalic astrocyte-derived neurotrophic factor).

7. Microparticle according to any of claims 1 to 6, wherein the angiogenic factor is selected from angiopoietin (Ang); basic fibroblast growth factor (bFGF / FGF2); beta transformer growth factor (TGFβ), alpha transformer growth factor (TGFα), placenta growth factor (PIGF); epidermal growth factor (EGF); vascular endothelial growth factor (VEGF); platelet-derived growth factor (PDGF); vasoactive intestinal polypeptide (VIP); hepatocyte growth factor (HGF), cardiotrophins, bone morphogenetic proteins (BMP) or sonic hedgehog (SHH).

8. Microparticle according to any one of claims 1 to 7, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and Huntington's disease.

9. Microparticle according to any one of claims 1 to 8, wherein the administration of the microparticle at the level of the cerebral cortex is carried out in a subdural or subarachnoid manner.

10. Microparticle according to any of claims 1 to 9, wherein the administration of the microparticle at the level of the cerebral cortex is carried out by bilateral craniotomy.